Antigen binding proteins that bind ErbB3

ABSTRACT

There is disclosed compositions and methods relating to or derived from anti-ErbB3 antibodies. More specifically, there is disclosed fully human antibodies that bind ErbB3, ErbB3-binding fragments and derivatives of such antibodies, and ErbB3-binding polypeptides comprising such fragments. Further still, there is disclosed nucleic acids encoding such antibodies, antibody fragments and derivatives and polypeptides, cells comprising such polynucleotides, methods of making such antibodies, antibody fragments and derivatives and polypeptides, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating or diagnosing subjects having ErbB3 related disorders or conditions, including various inflammatory disorders and various cancers.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims priority from U.S. provisional patentapplication 61/718,657 filed 25 Oct. 2012.

TECHNICAL FIELD

The present disclosure provides compositions and methods relating to orderived from anti-ErbB3 antibodies. More specifically, the presentdisclosure provides human antibodies that bind ErbB3, ErbB3-bindingfragments and derivatives of such antibodies, and ErbB3-bindingpolypeptides comprising such fragments. Further still, the presentdisclosure provides nucleic acids encoding such antibodies, antibodyfragments and derivatives and polypeptides, cells comprising suchpolynucleotides, methods of making such antibodies, antibody fragmentsand derivatives and polypeptides, and methods of using such antibodies,antibody fragments and derivatives and polypeptides, including methodsof treating or diagnosing subjects having ErbB3 related disorders orconditions, including various inflammatory disorders and variouscancers.

BACKGROUND

HER3/c-ErbB3 (referred to herein as ErbB3) is a member of the epidermalgrowth factor receptor (EGFR) family. ErbB3 binds neuregulin/heregulin(NRG/HRG). Receptors in the EGFR family are single transmembranereceptors with an intracellular tyrosine kinase domain. While the otherEGFR family members (such as EGFR/HER1/ErbB1, HER2/ErbB2, andHER4/ErbB4) each have tyrosine kinase activity, ErbB3 has little or notyrosine kinase activity, and thus is “kinase-dead.”

The extracellular domain (ECD) of the EGFR family contains four domains.Domains 1 and 3 (also known as domains L1 and L2) are responsible forligand binding. Cysteine-rich domains 2 and 4 (also known as domains C1and C2) are involved in dimerization with receptor partners. Upon ligandbinding, the ECD undergoes conformational changes. The interaction ofdomains 2 and 4, which maintains the tethered (inactive) conformation ofthe receptor, is relieved, and an extended (active) conformation isadopted. The extended conformation favors dimerization with otherreceptor partners. HER2/ErbB2 is the only exception to this generalrule, i.e., Her2-ECD is constitutively in the extended conformation.

Overexpression of ErbB3 is associated with poor prognosis in variouscarcinomas (e.g., breast, ovarian, prostate, colorectal, pancreatic,gastric, and head and neck cancers). Overexpression of ErbB3 alsocorrelates with local to distal metastasis in lung, gastric, andcolorectal cancers, and bone invasion in prostate cancer (Sithanandam etal., 2008, Cancer Gene Therapy 15:413). Overexpression of ErbB3 has beenlinked to resistance to several cancer treatments, including treatmentwith EGFR tyrosine kinase inhibitors in non-small cell lung cancer(NSCLC) and head and neck cancers, treatment with Her2 inhibitor inbreast cancers, and treatment with radiotherapy in pancreatic cancers.Moreover, overexpression of NRG, a ligand for ErbB3, was also linked toresistance to EGFR tyrosine kinase inhibitor treatment. Chen et al.describe the use of anti-ErbB3 monoclonal antibodies that inhibit NRGfunction and show growth inhibitory activity against breast and ovariancancer cells (Chen et al., 1996, J. Biol. Chem. 271: 7620).

ErbB3 has been found to be overexpressed in breast (Lemoine et al., Br.J. Cancer, 66:1116-1121 (1992)), gastrointestinal (Poller et al., J.Pathol., 168:275-280 (1992), Rajkumer et al., J. Pathol., 170:271-278(1993), and Sanidas et al., Int. J. Cancer, 54:935-940 (1993)), andpancreatic cancers (Lemoine et at, J. Pathol., 168:269-273 (1992), andFriess et al., Clinical Cancer Research, 1:1413-1420 (1995)).

Therefore, there is a need in the art for an improved anti-ErbB3antibodies that can be used as therapeutic agents.

SUMMARY

The present disclosure provides a fully human antibody of an IgG classthat binds to an ErbB3 epitope with a binding affinity of at least10⁻⁶M, which has a heavy chain variable domain sequence that is at least95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ IDNO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45,SEQ ID NO. 47, SEQ ID NO.49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO.55, SEQ ID NO. 57, SEQ ID NO. 59, and combinations thereof, and that hasa light chain variable domain sequence that is at least 95% identical tothe amino acid sequences selected from the group consisting of SEQ IDNO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ IDNO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30,SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO.40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ IDNO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQID NO. 60, and combinations thereof. Preferably, the fully humanantibody has both a heavy chain and a light chain wherein the antibodyhas a heavy chain/light chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1/SEQ ID NO. 2 (called A01 herein), SEQID NO. 3/SEQ ID NO. 4 (called A02 herein), SEQ ID NO. 5/SEQ ID NO. 6(called A04 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called A08 herein), SEQID NO. 9/SEQ ID NO. 10 (called A11 herein), SEQ ID NO. 11/SEQ ID NO. 12(called A12 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called B01 herein),SEQ ID NO. 15/SEQ ID NO. 16 (called B03 herein), SEQ ID NO. 17/SEQ IDNO. 18 (called B04 herein), SEQ ID NO. 19/SEQ ID NO. 20 (called B05herein), SEQ ID NO. 21/SEQ ID NO. 22 (called B08 herein), SEQ ID NO.23/SEQ ID NO. 24 (called B10 herein), SEQ ID NO. 25/SEQ ID NO. 26(called B11 herein), SEQ ID NO. 27/SEQ ID NO. 28 (called C03 herein),SEQ ID NO. 29/SEQ ID NO. 30 (called C07 herein), SEQ ID NO. 31/SEQ IDNO. 32 (called C10 herein), SEQ ID NO. 33/SEQ ID NO. 34 (called C12herein), SEQ ID NO. 35/SEQ ID NO. 36 (called D01 herein), SEQ ID NO.37/SEQ ID NO. 38 (called D03 herein), SEQ ID NO. 39/SEQ ID NO. 40(called D04 herein), SEQ ID NO. 41/SEQ ID NO. 42 (called D07 herein),SEQ ID NO. 43/SEQ ID NO. 44 (called D08 herein), SEQ ID NO. 45/SEQ IDNO. 46 (called D10 herein), SEQ ID NO. 47/SEQ ID NO. 48 (called D11herein), SEQ ID NO. 49/SEQ ID NO. 50 (called E04 herein), SEQ ID NO.51/SEQ ID NO. 52 (called E05 herein), SEQ ID NO. 53/SEQ ID NO. 54(called E08 herein), SEQ ID NO. 55/SEQ ID NO. 56 (called F10 herein),SEQ ID NO. 57/SEQ ID NO. 58 (called G01 herein), SEQ ID NO. 59/SEQ IDNO. 60 (called H06 herein), and combinations thereof.

The present disclosure provides a fully human antibody Fab fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.49, SEQ ID NO. 51, SEQ IDNO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, and combinationsthereof, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ IDNO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46,SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO.56, SEQ ID NO. 58, SEQ ID NO. 60, and combinations thereof. Preferably,the fully human antibody Fab fragment has both a heavy chain variabledomain region and a light chain variable domain region wherein theantibody has a heavy chain/light chain variable domain sequence selectedfrom the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ IDNO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ IDNO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ IDNO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ IDNO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ IDNO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ IDNO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ IDNO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ IDNO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ IDNO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ IDNO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ IDNO. 59/SEQ ID NO. 60, and combinations thereof.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connection the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, andcombinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ IDNO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, and combinationsthereof. Preferably, the fully human single chain antibody has both aheavy chain variable domain region and a light chain variable domainregion, wherein the single chain fully human antibody has a heavychain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ IDNO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ IDNO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ IDNO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ IDNO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ IDNO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ IDNO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ IDNO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ IDNO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ IDNO. 50, SEQ ID NO. 51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ IDNO. 55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ IDNO. 60, and combinations thereof.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers, comprising administering an effectiveamount of an anti-ErbB3 polypeptide, wherein the anti-ErbB3 polypeptideis selected from the group consisting of a fully human antibody of anIgG class that binds to an ErbB3 epitope with a binding affinity of atleast 10⁻⁶M, a fully human antibody Fab fragment, having a variabledomain region from a heavy chain and a variable domain region from alight chain, a single chain human antibody, having a variable domainregion from a heavy chain and a variable domain region from a lightchain and a peptide linker connection the heavy chain and light chainvariable domain regions, and combinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.49, SEQ ID NO. 51, SEQ IDNO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, and combinationsthereof, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ IDNO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46,SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO.56, SEQ ID NO. 58, SEQ ID NO. 60, and combinations thereof;

wherein the fully human antibody Fab fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ IDNO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.49, SEQID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59,and combinations thereof, and that has the light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ IDNO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, and combinationsthereof; and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, andcombinations thereof, and that has the light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ IDNO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, and combinationsthereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2(called A01 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called A02 herein), SEQID NO. 5/SEQ ID NO. 6 (called A04 herein), SEQ ID NO. 7/SEQ ID NO. 8(called A08 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called A11 herein), SEQID NO. 11/SEQ ID NO. 12 (called A12 herein), SEQ ID NO. 13/SEQ ID NO. 14(called B01 herein), SEQ ID NO. 15/SEQ ID NO. 16 (called B03 herein),SEQ ID NO. 17/SEQ ID NO. 18 (called B04 herein), SEQ ID NO. 19/SEQ IDNO. 20 (called B05 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called B08herein), SEQ ID NO. 23/SEQ ID NO. 24 (called B10 herein), SEQ ID NO.25/SEQ ID NO.26 (called B11 herein), SEQ ID NO. 27/SEQ ID NO. 28 (calledC03 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called C07 herein), SEQ ID NO.31/SEQ ID NO. 32 (called C10 herein), SEQ ID NO. 33/SEQ ID NO. 34(called C12 herein), SEQ ID NO. 35/SEQ ID NO. 36 (called D01 herein),SEQ ID NO. 37/SEQ ID NO. 38 (called D03 herein), SEQ ID NO. 39/SEQ IDNO. 40 (called D04 herein), SEQ ID NO. 41/SEQ ID NO. 42 (called D07herein), SEQ ID NO. 43/SEQ ID NO. 44 (called D08 herein), SEQ ID NO.45/SEQ ID NO. 46 (called D10 herein), SEQ ID NO. 47/SEQ ID NO. 48(called D11 herein), SEQ ID NO. 49/SEQ ID NO. 50 (called E04 herein),SEQ ID NO. 51/SEQ ID NO. 52 (called E05 herein), SEQ ID NO. 53/SEQ IDNO. 54 (called E08 herein), SEQ ID NO. 55/SEQ ID NO. 56 (called F10herein), SEQ ID NO. 57/SEQ ID NO. 58 (called G01 herein), SEQ ID NO.59/SEQ ID NO. 60 (called H06 herein), and combinations thereof.Preferably, the fully human antibody Fab fragment has both a heavy chainvariable domain region and a light chain variable domain region whereinthe antibody has a heavy chain/light chain variable domain sequenceselected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2 (calledA01 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called A02 herein), SEQ ID NO.5/SEQ ID NO. 6 (called A04 herein), SEQ ID NO. 7/SEQ ID NO. 8 (calledA08 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called A11 herein), SEQ ID NO.11/SEQ ID NO. 12 (called A12 herein), SEQ ID NO. 13/SEQ ID NO. 14(called B01 herein), SEQ ID NO. 15/SEQ ID NO. 16 (called B03 herein),SEQ ID NO. 17/SEQ ID NO. 18 (called B04 herein), SEQ ID NO. 19/SEQ IDNO. 20 (called B05 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called B08herein), SEQ ID NO. 23/SEQ ID NO. 24 (called B10 herein), SEQ ID NO.25/SEQ ID NO.26 (called B11 herein), SEQ ID NO. 27/SEQ ID NO. 28 (calledC03 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called C07 herein), SEQ ID NO.31/SEQ ID NO. 32 (called C10 herein), SEQ ID NO. 33/SEQ ID NO. 34(called C12 herein), SEQ ID NO. 35/SEQ ID NO. 36 (called D01 herein),SEQ ID NO. 37/SEQ ID NO. 38 (called D03 herein), SEQ ID NO. 39/SEQ IDNO. 40 (called D04 herein), SEQ ID NO. 41/SEQ ID NO. 42 (called D07herein), SEQ ID NO. 43/SEQ ID NO. 44 (called D08 herein), SEQ ID NO.45/SEQ ID NO. 46 (called D10 herein), SEQ ID NO. 47/SEQ ID NO. 48(called D11 herein), SEQ ID NO. 49/SEQ ID NO. 50 (called E04 herein),SEQ ID NO. 51/SEQ ID NO. 52 (called E05 herein), SEQ ID NO. 53/SEQ IDNO. 54 (called E08 herein), SEQ ID NO. 55/SEQ ID NO. 56 (called F10herein), SEQ ID NO. 57/SEQ ID NO. 58 (called G01 herein), SEQ ID NO.59/SEQ ID NO. 60 (called H06 herein), and combinations thereof.Preferably, the fully human single chain antibody has both a heavy chainvariable domain region and a light chain variable domain region, whereinthe single chain fully human antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO.26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO.31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO.36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO.41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO.46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO.51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO.56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, andcombinations thereof.

Preferably, the broad spectrum of mammalian cancers to be treated thecancer is an ErbB3-positive cancer. Preferably, the broad spectrum ofmammalian cancers to be treated is selected from the group consisting ofcarcinomas, prostate cancer, non-small cell lung cancer, breast cancer,endometrial cancer, ovarian cancer, gastric cancers, head and neckcancers, and colon cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the binding of anti-ErbB3 antibodies to Human recombinantErbB3 Protein, as measured by ELISA. Recombinant Human Proteins EGFR andErbB2, other members of the ErbB Family, were used as controls. Theresults show that anti-ErbB3 antibodies bind specifically to ErbB3.

FIG. 1B shows the results of a Mouse Cross Reactivity ELISA test. Thestrong signal detected shows that most of anti-Human ErbB3 antibodiesalso bind to Mouse recombinant ErbB3 Protein.

FIG. 2 shows the binding of exemplary anti-ErbB3 Antibodies to HumanErbB3 expressed at the Cell Surface (MCF7), as analyzed by FlowCytometry.

FIG. 3 is a HPLC-ANSEC analysis of selected antibodies as shown.

FIG. 4 shows the inhibition of Heregulin-mediated MCF7 proliferation.Cells were incubated for 3 days with Heregulin in the presence orabsence of anti-ErbB3 antibodies. Cell proliferation was then analyzedby MTT assay. The graph shows the dose curves obtained for selectedantibodies. Under the graph is a table with the IC₅₀ values obtained forthose antibodies.

FIG. 5 shows that anti-ErB3 antibodies inhibit Her3 Tyrosinephosphorylation in a dose dependent manner, as measured by ELISA.

FIG. 6 shows that anti-ErB3 antibodies inhibit p42/44 MAP kinasephosphorylation in a dose dependent manner, as measured by ELISA.

FIG. 7 shows that exemplary anti-ErbB3 antibodies block ErbB3 binding toimmobilized Heregulin, as measured by OCTET. The absence of a bindingsignal reflects a strong inhibition of ErB3 binding to its ligand.

FIG. 8 is a picture obtained by fluorescence microscopy analysis,showing that ErbB3-A04 antibody can induce ErbB3 ReceptorInternalization in MCF7.

FIG. 9 shows the ability of ErbB3-A04 antibody to inhibit MCF7migration. Data were measured using non-invasive real-time Impedancemeasurements with an xCELLigence RTCA MP system (Roche).

FIG. 10 shows a synergetic inhibitory effect observed when exemplaryanti-ErbB3 and anti-EGFR antibodies are used in combination in an assaymeasuring A431NS proliferation.

FIG. 11 shows the synergetic inhibitory effect observed when exemplaryanti-ErbB3 and either anti-EGFR or anti-Her2 antibodies are used incombination in an assay measuring a) MAPK phosphorylation and b) Aktphosphorylation in A431NS, following different stimulations (NRGb1 only,FBS only, or NRGb1+FBS).

DETAILED DESCRIPTION

The present disclosure provides a fully human antibody of an IgG classthat binds to an ErbB3 epitope with a binding affinity of 10⁻⁶M or less,that has a heavy chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ IDNO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQID NO. 39, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47,SEQ ID NO.49, SEQ ID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO.57, SEQ ID NO. 59, and combinations thereof, and that has a light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ IDNO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60,and combinations thereof. Preferably, the fully human antibody has botha heavy chain and a light chain wherein the antibody has a heavychain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2 (called A01 herein), SEQ ID NO.3/SEQ ID NO. 4 (called A02 herein), SEQ ID NO. 5/SEQ ID NO. 6 (calledA04 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called A08 herein), SEQ ID NO.9/SEQ ID NO. 10 (called A11 herein), SEQ ID NO. 11/SEQ ID NO. 12 (calledA12 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called B01 herein), SEQ ID NO.15/SEQ ID NO. 16 (called B03 herein), SEQ ID NO. 17/SEQ ID NO. 18(called B04 herein), SEQ ID NO. 19/SEQ ID NO. 20 (called B05 herein),SEQ ID NO. 21/SEQ ID NO. 22 (called B08 herein), SEQ ID NO. 23/SEQ IDNO. 24 (called B10 herein), SEQ ID NO. 25/SEQ ID NO.26 (called B11herein), SEQ ID NO. 27/SEQ ID NO. 28 (called C03 herein), SEQ ID NO.29/SEQ ID NO. 30 (called C07 herein), SEQ ID NO. 31/SEQ ID NO. 32(called C10 herein), SEQ ID NO. 33/SEQ ID NO. 34 (called C12 herein),SEQ ID NO. 35/SEQ ID NO. 36 (called D01 herein), SEQ ID NO. 37/SEQ IDNO. 38 (called D03 herein), SEQ ID NO. 39/SEQ ID NO. 40 (called D04herein), SEQ ID NO. 41/SEQ ID NO. 42 (called D07 herein), SEQ ID NO.43/SEQ ID NO. 44 (called D08 herein), SEQ ID NO. 45/SEQ ID NO. 46(called D10 herein), SEQ ID NO. 47/SEQ ID NO. 48 (called D11 herein),SEQ ID NO. 49/SEQ ID NO. 50 (called E04 herein), SEQ ID NO. 51/SEQ IDNO. 52 (called E05 herein), SEQ ID NO. 53/SEQ ID NO. 54 (called E08herein), SEQ ID NO. 55/SEQ ID NO. 56 (called F10 herein), SEQ ID NO.57/SEQ ID NO. 58 (called G01 herein), SEQ ID NO. 59/SEQ ID NO. 60(called H06 herein), and combinations thereof.

The present disclosure provides a fully human antibody Fab fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.49, SEQ ID NO. 51, SEQ IDNO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, and combinationsthereof, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ IDNO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46,SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO.56, SEQ ID NO. 58, SEQ ID NO. 60, and combinations thereof. Preferably,the fully human antibody Fab fragment has both a heavy chain variabledomain region and a light chain variable domain region wherein theantibody has a heavy chain/light chain variable domain sequence selectedfrom the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ IDNO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ IDNO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ IDNO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ IDNO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ IDNO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ IDNO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ IDNO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ IDNO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ IDNO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ IDNO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ IDNO. 59/SEQ ID NO. 60, and combinations thereof.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connection the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, andcombinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ IDNO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, and combinationsthereof. Preferably, the fully human single chain antibody has both aheavy chain variable domain region and a light chain variable domainregion, wherein the single chain fully human antibody has a heavychain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ IDNO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ IDNO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ IDNO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ IDNO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ IDNO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ IDNO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ IDNO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ IDNO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ IDNO. 50, SEQ ID NO. 51/SEQ ID NO. 52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ IDNO. 55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ IDNO. 60, and combinations thereof.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers or inflammatory diseases or autoimmunediseases, comprising administering an effective amount of an anti-ErbB3polypeptide, wherein the anti-ErbB3 polypeptide is selected from thegroup consisting of a fully human antibody of an IgG class that binds toan ErbB3 epitope with a binding affinity of at least 10⁻⁶M, a fullyhuman antibody Fab fragment, having a variable domain region from aheavy chain and a variable domain region from a light chain, a singlechain human antibody, having a variable domain region from a heavy chainand a variable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions, andcombinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.49, SEQ ID NO. 51, SEQ IDNO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, and combinationsthereof, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ IDNO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46,SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO.56, SEQ ID NO. 58, SEQ ID NO. 60, and combinations thereof;

wherein the fully human antibody Fab fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ IDNO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.49, SEQID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59,and combinations thereof, and that has the light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ IDNO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, and combinationsthereof; and wherein the single chain human antibody has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ IDNO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO.49, SEQID NO. 51, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59,and combinations thereof, and that has the light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ IDNO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, and combinationsthereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO.42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO.47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO.52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO.57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, and combinations thereof.Preferably, the fully human antibody Fab fragment has both a heavy chainvariable domain region and a light chain variable domain region whereinthe antibody has a heavy chain/light chain variable domain sequenceselected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ IDNO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO.8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO.13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO.18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO.23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO.28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO.33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO.38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO. 42, SEQ ID NO.43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO. 47/SEQ ID NO.48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO. 52, SEQ ID NO.53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO. 57/SEQ ID NO.58, SEQ ID NO. 59/SEQ ID NO. 60, and combinations thereof. Preferably,the fully human single chain antibody has both a heavy chain variabledomain region and a light chain variable domain region, wherein thesingle chain fully human antibody has a heavy chain/light chain variabledomain sequence selected from the group consisting of SEQ ID NO. 1/SEQID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ IDNO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO.12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 41/SEQ ID NO.42, SEQ ID NO. 43/SEQ ID NO. 44, SEQ ID NO. 45/SEQ ID NO. 46, SEQ ID NO.47/SEQ ID NO. 48, SEQ ID NO. 49/SEQ ID NO. 50, SEQ ID NO. 51/SEQ ID NO.52, SEQ ID NO. 53/SEQ ID NO. 54, SEQ ID NO. 55/SEQ ID NO. 56, SEQ ID NO.57/SEQ ID NO. 58, SEQ ID NO. 59/SEQ ID NO. 60, and combinations thereof.

Preferably, the broad spectrum of mammalian cancers to be treated thecancer is an ErbB3-positive cancer. Preferably, the broad spectrum ofmammalian cancers to be treated is selected from the group consisting ofcarcinomas, prostate cancer, non-small cell lung cancer, breast cancer,endometrial cancer, ovarian cancer, gastric cancers, head and neckcancers, and colon cancer.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.

The variable regions of naturally occurring immunoglobulin chainsexhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarity determining regions or CDRs. From N-terminus toC-terminus, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat et al. inSequences of Proteins of Immunological Interest, 5^(th) Ed., US Dept. ofHealth and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.Other numbering systems for the amino acids in immunoglobulin chainsinclude IMGT.RTM. (international ImMunoGeneTics information system;Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honeggerand Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

Antibodies can be obtained from sources such as serum or plasma thatcontain immunoglobulins having varied antigenic specificity. If suchantibodies are subjected to affinity purification, they can be enrichedfor a particular antigenic specificity. Such enriched preparations ofantibodies usually are made of less than about 10% antibody havingspecific binding activity for the particular antigen. Subjecting thesepreparations to several rounds of affinity purification can increase theproportion of antibody having specific binding activity for the antigen.Antibodies prepared in this manner are often referred to as“monospecific.” Monospecfic antibody preparations can be made up ofabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,99%, or 99.9% antibody having specific binding activity for theparticular antigen.

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion thereof that competes with the intact antibody forspecific binding, unless otherwise specified. Antigen binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),and complementarity determining region (CDR) fragments, single-chainantibodies (scFv), chimeric antibodies, diabodies, triabodies,tetrabodies, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H1) domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H1) domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or VL domain (U.S. Pat. Nos. 6,846,634; 6,696,245, US PatentApplications 2002/02512; 2004/0202995; 2004/0038291; 2004/0009507;2003/0039958, and Ward et al., Nature 341:544-546, 1989).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (Bird et al., 1988, Science242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-83). Diabodies are bivalent antibodies comprising twopolypeptide chains, wherein each polypeptide chain comprises V_(H) andV_(L) domains joined by a linker that is too short to allow for pairingbetween two domains on the same chain, thus allowing each domain to pairwith a complementary domain on another polypeptide chain (Holliger etal., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al.,1994, Structure 2:1121-23). If the two polypeptide chains of a diabodyare identical, then a diabody resulting from their pairing will have twoidentical antigen binding sites. Polypeptide chains having differentsequences can be used to make a diabody with two different antigenbinding sites. Similarly, tribodies and tetrabodies are antibodiescomprising three and four polypeptide chains, respectively, and formingthree and four antigen binding sites, respectively, which can be thesame or different.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. supra; Lefranc et al., supra and/or Honegger and Pluckthun,supra. One or more CDRs may be incorporated into a molecule eithercovalently or noncovalently to make it an antigen binding protein. Anantigen binding protein may incorporate the CDR(s) as part of a largerpolypeptide chain, may covalently link the CDR(s) to another polypeptidechain, or may incorporate the CDR(s) noncovalently. The CDRs permit theantigen binding protein to specifically bind to a particular antigen ofinterest.

An antigen binding protein may have one or more binding sites. If thereis more than one binding site, the binding sites may be identical to oneanother or may be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

A humanized antibody has a sequence that differs from the sequence of anantibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one embodiment, one or more of the CDRs are derivedfrom a human anti-ErbB3 antibody. In another embodiment, all of the CDRsare derived from a human anti-ErbB3 antibody. In another embodiment, theCDRs from more than one human anti-ErbB3 antibodies are mixed andmatched in a chimeric antibody. For instance, a chimeric antibody maycomprise a CDR1 from the light chain of a first human anti-PAR-2antibody, a CDR2 and a CDR3 from the light chain of a second humananti-ErbB3 antibody, and the CDRs from the heavy chain from a thirdanti-ErbB3 antibody. Other combinations are possible.

Further, the framework regions may be derived from one of the sameanti-ErbB3 antibodies, from one or more different antibodies, such as ahuman antibody, or from a humanized antibody. In one example of achimeric antibody, a portion of the heavy and/or light chain isidentical with, homologous to, or derived from an antibody from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with,homologous to, or derived from an antibody (-ies) from another speciesor belonging to another antibody class or subclass. Also included arefragments of such antibodies that exhibit the desired biologicalactivity (i.e., the ability to specifically bind ErbB3).

A “neutralizing antibody” or an “inhibitory antibody” is an antibodythat inhibits the activation of ErbB3 when an excess of the anti-ErbB3antibody reduces the amount of activation by at least about 20% using anassay such as those described herein in the Examples. In variousembodiments, the antigen binding protein reduces the amount of amount ofactivation of ErbB3 by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 99%, and 99.9%.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specificationand using techniques known in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function. Methods to identify protein sequences that fold into aknown three-dimensional structure are known. See, Bowie et al., 1991,Science 253:164.

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human ErbB3) if it binds to the antigen with a dissociation constant of1 nanomolar or less.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent identity” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragment,derivative, mutein, or variant thereof.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (see Rasmussen et al.,1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient inDHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20),HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derivedfrom the African green monkey kidney cell line CV1 (ATCC CCL 70) (seeMcMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cellssuch as 293,293 EBNA or MSR 293, human epidermal A431 cells, humanColo205 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a hostcell is a cultured cell that can be transformed or transfected with apolypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

Preferably, the mammalian cancer to be treated is selected from thegroup consisting of ovarian, colon, breast or hepatic carcinoma celllines, myelomas, neuroblastic-derived CNS tumors, monocytic leukemias,B-cell derived leukemia's, T-cell derived leukemias, B-cell derivedlymphomas, T-cell derived lymphomas, mast cell derived tumors, andcombinations thereof.

Polypeptides of the present disclosure can be produced using anystandard methods known in the art. In one example, the polypeptides areproduced by recombinant DNA methods by inserting a nucleic acid sequence(e.g., a cDNA) encoding the polypeptide into a recombinant expressionvector and expressing the DNA sequence under conditions promotingexpression.

Nucleic acids encoding any of the various polypeptides disclosed hereinmay be synthesized chemically. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells. See for example: Mayfield et al., Proc.Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr.Purif. 2002 (1):96-105; Connell N D. Curr. Opin. Biotechnol. 200112(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38; andSharp et al. Yeast. 1991 7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants is additionally incorporated.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).

The expression construct is introduced into the host cell using a methodappropriate to the host cell. A variety of methods for introducingnucleic acids into host cells are known in the art, including, but notlimited to, electroporation; transfection employing calcium chloride,rubidium chloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent). Suitable host cells include prokaryotes,yeast, mammalian cells, or bacterial cells.

Suitable bacteria include gram negative or gram positive organisms, forexample, E. coli or Bacillus spp. Yeast, preferably from theSaccharomyces species, such as S. cerevisiae, may also be used forproduction of polypeptides. Various mammalian or insect cell culturesystems can also be employed to express recombinant proteins.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, (Bio/Technology, 6:47, 1988).Examples of suitable mammalian host cell lines include endothelialcells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinesehamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, andBHK cell lines. Purified polypeptides are prepared by culturing suitablehost/vector systems to express the recombinant proteins. For manyapplications, the small size of many of the polypeptides disclosedherein would make expression in E. coli as the preferred method forexpression. The protein is then purified from culture media or cellextracts.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system).

ErbB3-binding polypeptides can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The polypeptides of the present disclosure can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product.

Post-Translational Modifications of Polypeptides

In certain embodiments, the binding polypeptides of the invention mayfurther comprise post-translational modifications. Exemplarypost-translational protein modifications include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified soluble polypeptides may contain non-amino acid elements, suchas lipids, poly- or mono-saccharide, and phosphates. A preferred form ofglycosylation is sialylation, which conjugates one or more sialic acidmoieties to the polypeptide. Sialic acid moieties improve solubility andserum half-life while also reducing the possible immunogeneticity of theprotein. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.

In one specific embodiment, modified forms of the subject solublepolypeptides comprise linking the subject soluble polypeptides tononproteinaceous polymers. In one specific embodiment, the polymer ispolyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes,in the manner as set forth in U.S. Pat. No. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337. Examples of the modifiedpolypeptide include PEGylated VK-B8.

PEG is a water soluble polymer that is commercially available or can beprepared by ring-opening polymerization of ethylene glycol according tomethods well known in the art (Sandler and Karo, Polymer Synthesis,Academic Press, New York, Vol. 3, pages 138-161). The term “PEG” is usedbroadly to encompass any polyethylene glycol molecule, without regard tosize or to modification at an end of the PEG, and can be represented bythe formula: X—O(CH₂CH₂O)_(n)-1CH₂CH₂OH (1), where n is 20 to 2300 and Xis H or a terminal modification, e.g., a C₁₋₄ alkyl. In one embodiment,the PEG of the invention terminates on one end with hydroxy or methoxy,i.e., X is H or CH₃ (“methoxy PEG”). A PEG can contain further chemicalgroups which are necessary for binding reactions; which results from thechemical synthesis of the molecule; or which is a spacer for optimaldistance of parts of the molecule. In addition, such a PEG can consistof one or more PEG side-chains which are linked together. PEGs with morethan one PEG chain are called multiarmed or branched PEGs. Branched PEGscan be prepared, for example, by the addition of polyethylene oxide tovarious polyols, including glycerol, pentaerythriol, and sorbitol. Forexample, a four-armed branched PEG can be prepared from pentaerythrioland ethylene oxide. Branched PEG are described in, for example, EP-A 0473 084 and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEGside-chains (PEG2) linked via the primary amino groups of a lysine(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski et al., J. Biol. Chem., 252, 3571 (1977) and J. Biol. Chem.,252, 3582 (1977), Zalipsky, et al., and Harris et. al., in:Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22). It is noted that a binding polypeptide containing a PEGmolecule is also known as a conjugated protein, whereas the proteinlacking an attached PEG molecule can be referred to as unconjugated.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated binding polypeptide, such as size exclusion(e.g. gel filtration) and ion exchange chromatography. Products may alsobe separated using SDS-PAGE. Products that may be separated includemono-, di-, tri-poly- and un-pegylated binding polypeptide, as well asfree PEG. The percentage of mono-PEG conjugates can be controlled bypooling broader fractions around the elution peak to increase thepercentage of mono-PEG in the composition. About ninety percent mono-PEGconjugates represents a good balance of yield and activity. Compositionsin which, for example, at least ninety-two percent or at leastninety-six percent of the conjugates are mono-PEG species may bedesired. In an embodiment of this invention the percentage of mono-PEGconjugates is from ninety percent to ninety-six percent.

In one embodiment, PEGylated binding polypeptide of the inventioncontain one, two or more PEG moieties. In one embodiment, the PEGmoiety(ies) are bound to an amino acid residue which is on the surfaceof the protein and/or away from the surface that contacts the targetligand. In one embodiment, the combined or total molecular mass of PEGin PEG-binding polypeptide is from about 3,000 Da to 60,000 Da,optionally from about 10,000 Da to 36,000 Da. In a one embodiment, thePEG in pegylated binding polypeptide is a substantially linear,straight-chain PEG.

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t_(1/2)) which isenhanced relative to the half-life of the unmodified protein. Thehalf-life of PEG-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified binding polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

Therapeutic Formulations and Modes of Administration

The present disclosure features methods for treating conditions orpreventing pre-conditions which respond to an inhibition of ErbB3biological activity. Preferred examples are conditions that arecharacterized by inflammation or cellular hyperproliferation. Techniquesand dosages for administration vary depending on the type of specificpolypeptide and the specific condition being treated but can be readilydetermined by the skilled artisan. In general, regulatory agenciesrequire that a protein reagent to be used as a therapeutic is formulatedso as to have acceptably low levels of pyrogens. Accordingly,therapeutic formulations will generally be distinguished from otherformulations in that they are substantially pyrogen free, or at leastcontain no more than acceptable levels of pyrogen as determined by theappropriate regulatory agency (e.g., FDA).

Therapeutic compositions of the present disclosure may be administeredwith a pharmaceutically acceptable diluent, carrier, or excipient, inunit dosage form. Administration may be parenteral (e.g., intravenous,subcutaneous), oral, or topical, as non-limiting examples. In addition,any gene therapy technique, using nucleic acids encoding thepolypeptides of the invention, may be employed, such as naked DNAdelivery, recombinant genes and vectors, cell-based delivery, includingex vivo manipulation of patients' cells, and the like.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration; or a liquid forintravenous, subcutaneous or parenteral administration; gel, lotion,ointment, cream, or a polymer or other sustained release vehicle forlocal administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

The polypeptide may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andanti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the polypeptideis administered at about 0.01 μg/kg to about 50 mg/kg per day,preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1mg/kg to about 20 mg/kg per day. The polypeptide may be given daily(e.g., once, twice, three times, or four times daily) or preferably lessfrequently (e.g., weekly, every two weeks, every three weeks, monthly,or quarterly). In addition, as is known in the art, adjustments for ageas well as the body weight, general health, sex, diet, time ofadministration, drug interaction, and the severity of the disease may benecessary, and will be ascertainable with routine experimentation bythose skilled in the art.

Exemplary Uses

The ErbB3 binding proteins described herein and their related variantsare useful in a number of therapeutic and diagnostic applications. Theseinclude the inhibition of the biological activity of ErbB3 by competingfor or blocking the binding to an ErbB3 as well as the delivery ofcytotoxic or imaging moieties to cells, preferably cells expressingErbB3. The small size and stable structure of these molecules can beparticularly valuable with respect to manufacturing of the drug, rapidclearance from the body for certain applications where rapid clearanceis desired or formulation into novel delivery systems that are suitableor improved using a molecule with such characteristics.

On the basis of their efficacy as inhibitors of ErbB3 biologicalactivity, the polypeptides of this disclosure are effective against anumber of cancer conditions as well as complications arising fromcancer, such as pleural effusion and ascites, colorectal cancers, headand neck cancers, small cell lung cancer, non-small cell lung cancer(NSCLC) and pancreatic cancer. Non-limiting examples of cancers includebladder, blood, bone, brain, breast, cartilage, colon kidney, liver,lung, lymph node, nervous tissue, ovary, pancreatic, prostate, skeletalmuscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid,trachea, urogenital tract, ureter, urethra, uterus, or vaginal cancer.

An ErbB3 binding polypeptide can be administered alone or in combinationwith one or more additional therapies such as chemotherapy radiotherapy,immunotherapy, surgical intervention, or any combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above. Preferably,in breast cancer the combination is with Herceptin or other Her-2therapies.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject anti-ErbB3 antibodies agents of theinvention can be used alone. Alternatively, the subject agents may beused in combination with other conventional anti-cancer therapeuticapproaches directed to treatment or prevention of proliferativedisorders (e.g., tumor). For example, such methods can be used inprophylactic cancer prevention, prevention of cancer recurrence andmetastases after surgery, and as an adjuvant of other conventionalcancer therapy. The present disclosure recognizes that the effectivenessof conventional cancer therapies (e.g., chemotherapy, radiation therapy,phototherapy, immunotherapy, and surgery) can be enhanced through theuse of a subject polypeptide therapeutic agent.

A wide array of conventional compounds have been shown to haveanti-neoplastic activities. These compounds have been used aspharmaceutical agents in chemotherapy to shrink solid tumors, preventmetastases and further growth, or decrease the number of malignant cellsin leukemic or bone marrow malignancies. Although chemotherapy has beeneffective in treating various types of malignancies, manyanti-neoplastic compounds induce undesirable side effects. It has beenshown that when two or more different treatments are combined, thetreatments may work synergistically and allow reduction of dosage ofeach of the treatments, thereby reducing the detrimental side effectsexerted by each compound at higher dosages. In other instances,malignancies that are refractory to a treatment may respond to acombination therapy of two or more different treatments.

When a polypeptide therapeutic agent of the present invention isadministered in combination with another conventional anti-neoplasticagent, either concomitantly or sequentially, such therapeutic agent maybe found to enhance the therapeutic effect of the anti-neoplastic agentor overcome cellular resistance to such anti-neoplastic agent. Thisallows decrease of dosage of an anti-neoplastic agent, thereby reducingthe undesirable side effects, or restores the effectiveness of ananti-neoplastic agent in resistant cells.

Pharmaceutical compounds that may be used for combinatory anti-tumortherapy include, merely to illustrate: aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

Certain chemotherapeutic anti-tumor compounds may be categorized bytheir mechanism of action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes—dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (e.g., VEGF inhibitors, fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),corticosteroids (cortisone, dexamethasone, hydrocortisone,methylpednisolone, prednisone, and prenisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; and chromatin disruptors.

Depending on the nature of the combinatory therapy, administration ofthe polypeptide therapeutic agents may be continued while the othertherapy is being administered and/or thereafter. Administration of thepolypeptide therapeutic agents may be made in a single dose, or inmultiple doses. In some instances, administration of the polypeptidetherapeutic agents is commenced at least several days prior to theconventional therapy, while in other instances, administration is beguneither immediately before or at the time of the administration of theconventional therapy.

In one example of a diagnostic application, a biological sample, such asserum or a tissue biopsy, from a patient suspected of having a conditioncharacterized by inappropriate angiogenesis is contacted with adetectably labeled polypeptide of the disclosure to detect levels ofErbB3. The levels of ErbB3 detected are then compared to levels of ErbB3detected in a normal sample also contacted with the labeled polypeptide.An increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%in the levels of the ErbB3 may be considered a diagnostic indicator.

In certain embodiments, the ErbB3 binding polypeptides are furtherattached to a label that is able to be detected (e.g., the label can bea radioisotope, fluorescent compound, enzyme or enzyme co-factor). Theactive moiety may be a radioactive agent, such as: radioactive heavymetals such as iron chelates, radioactive chelates of gadolinium ormanganese, positron emitters of oxygen, nitrogen, iron, carbon, orgallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ¹²³I, ¹²⁵I, ¹³¹I, ¹³²I, or⁹⁹Tc. A binding agent affixed to such a moiety may be used as an imagingagent and is administered in an amount effective for diagnostic use in amammal such as a human and the localization and accumulation of theimaging agent is then detected. The localization and accumulation of theimaging agent may be detected by radioscintigraphy, nuclear magneticresonance imaging, computed tomography or positron emission tomography.Immunoscintigraphy using ErbB3 binding polypeptides directed at ErbB3may be used to detect and/or diagnose cancers and vasculature. Forexample, any of the binding polypeptide against an ErbB3 marker labeledwith ⁹⁹Technetium, ¹¹¹Indium, or ¹²⁵Iodine may be effectively used forsuch imaging. As will be evident to the skilled artisan, the amount ofradioisotope to be administered is dependent upon the radioisotope.Those having ordinary skill in the art can readily formulate the amountof the imaging agent to be administered based upon the specific activityand energy of a given radionuclide used as the active moiety. Typically0.1-100 millicuries per dose of imaging agent, preferably 1-10millicuries, most often 2-5 millicuries are administered. Thus,compositions according to the present invention useful as imaging agentscomprising a targeting moiety conjugated to a radioactive moietycomprise 0.1-100 millicuries, in some embodiments preferably 1-10millicuries, in some embodiments preferably 2-5 millicuries, in someembodiments more preferably 1-5 millicuries.

The ErbB3 binding polypeptides can also be used to deliver additionaltherapeutic agents (including but not limited to drug compounds,chemotherapeutic compounds, and radiotherapeutic compounds) to a cell ortissue expressing ErbB3. In one example, the ErbB3 binding polypeptideis fused to a chemotherapeutic agent for targeted delivery of thechemotherapeutic agent to a tumor cell or tissue expressing ErbB3.

The ErbB3 binding polypeptides are useful in a variety of applications,including research, diagnostic and therapeutic applications. Forinstance, they can be used to isolate and/or purify receptor or portionsthereof, and to study receptor structure (e.g., conformation) andfunction.

In certain aspects, the various binding polypeptides can be used todetect or measure the expression of ErbB3, for example, on endothelialcells (e.g., venous endothelial cells), or on cells transfected with anErbB3 gene. Thus, they also have utility in applications such as cellsorting and imaging (e.g., flow cytometry, and fluorescence activatedcell sorting), for diagnostic or research purposes.

In certain embodiments, the binding polypeptides of fragments thereofcan be labeled or unlabeled for diagnostic purposes. Typically,diagnostic assays entail detecting the formation of a complex resultingfrom the binding of a binding polypeptide to ErbB3. The bindingpolypeptides or fragments can be directly labeled, similar toantibodies. A variety of labels can be employed, including, but notlimited to, radionuclides, fluorescers, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and4,098,876). When unlabeled, the binding polypeptides can be used inassays, such as agglutination assays. Unlabeled binding polypeptides canalso be used in combination with another (one or more) suitable reagentwhich can be used to detect the binding polypeptide, such as a labeledantibody reactive with the binding polypeptide or other suitable reagent(e.g., labeled protein A).

In one embodiment, the binding polypeptides of the present invention canbe utilized in enzyme immunoassays, wherein the subject polypeptides areconjugated to an enzyme. When a biological sample comprising an ErbB3protein is combined with the subject binding polypeptides, bindingoccurs between the binding polypeptides and the ErbB3 protein. In oneembodiment, a sample containing cells expressing an ErbB3 protein (e.g.,endothelial cells) is combined with the subject antibodies, and bindingoccurs between the binding polypeptides and cells bearing an ErbB3protein recognized by the binding polypeptide. These bound cells can beseparated from unbound reagents and the presence of the bindingpolypeptide-enzyme conjugate specifically bound to the cells can bedetermined, for example, by contacting the sample with a substrate ofthe enzyme which produces a color or other detectable change when actedon by the enzyme. In another embodiment, the subject bindingpolypeptides can be unlabeled, and a second, labeled polypeptide (e.g.,an antibody) can be added which recognizes the subject bindingpolypeptide.

In certain aspects, kits for use in detecting the presence of an ErbB3protein in a biological sample can also be prepared. Such kits willinclude an ErbB3 binding polypeptide which binds to an ErbB3 protein orportion of said receptor, as well as one or more ancillary reagentssuitable for detecting the presence of a complex between the bindingpolypeptide and the receptor protein or portions thereof. Thepolypeptide compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The binding polypeptides and/orantibodies, which can be labeled or unlabeled, can be included in thekits with adjunct ingredients (e.g., buffers, such as Tris, phosphateand carbonate, stabilizers, excipients, biocides and/or inert proteins,e.g., bovine serum albumin). For example, the binding polypeptidesand/or antibodies can be provided as a lyophilized mixture with theadjunct ingredients, or the adjunct ingredients can be separatelyprovided for combination by the user. Generally these adjunct materialswill be present in less than about 5% weight based on the amount ofactive binding polypeptide or antibody, and usually will be present in atotal amount of at least about 0.001% weight based on polypeptide orantibody concentration. Where a second antibody capable of binding tothe binding polypeptide is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above.

Similarly, the present disclosure also provides a method of detectingand/or quantitating expression of ErbB3, wherein a compositioncomprising a cell or fraction thereof (e.g., membrane fraction) iscontacted with a binding polypeptide which binds to an ErbB3 or portionof the receptor under conditions appropriate for binding thereto, andthe binding is monitored. Detection of the binding polypeptide,indicative of the formation of a complex between binding polypeptide andErbB3 or a portion thereof, indicates the presence of the receptor.Binding of a polypeptide to the cell can be determined by standardmethods, such as those described in the working examples. The method canbe used to detect expression of ErbB3 on cells from an individual.Optionally, a quantitative expression of ErbB3 on the surface ofendothelial cells can be evaluated, for instance, by flow cytometry, andthe staining intensity can be correlated with disease susceptibility,progression or risk.

The present disclosure also provides a method of detecting thesusceptibility of a mammal to certain diseases. To illustrate, themethod can be used to detect the susceptibility of a mammal to diseaseswhich progress based on the amount of ErbB3 present on cells and/or thenumber of ErbB3-positive cells in a mammal.

Polypeptide sequences are indicated using standard one- or three-letterabbreviations. Unless otherwise indicated, each polypeptide sequence hasamino termini at the left and a carboxy termini at the right; eachsingle-stranded nucleic acid sequence, and the top strand of eachdouble-stranded nucleic acid sequence, has a 5′ termini at the left anda 3′ termini at the right. A particular polypeptide sequence also can bedescribed by explaining how it differs from a reference sequence.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The terms “ErbB3 inhibitor” and “ErbB3 antagonist” are usedinterchangeably. Each is a molecule that detectably inhibits at leastone function of ErbB3. Conversely, an “ErbB3 agonist” is a molecule thatdetectably increases at least one function of ErbB3. The inhibitioncaused by an ErbB3 inhibitor need not be complete so long as it isdetectable using an assay. Any assay of a function of ErbB3 can be used,examples of which are provided herein. Examples of functions of ErbB3that can be inhibited by an ErbB3 inhibitor, or increased by an ErbB3agonist, include cancer cell growth or apoptosis (programmed celldeath), and so on. Examples of types of ErbB3 inhibitors and ErbB3agonists include, but are not limited to, ErbB3 binding polypeptidessuch as antigen binding proteins (e.g., ErbB3 inhibiting antigen bindingproteins), antibodies, antibody fragments, and antibody derivatives.

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric.

A “variant” of a polypeptide (for example, an antibody) comprises anamino acid sequence wherein one or more amino acid residues are insertedinto, deleted from and/or substituted into the amino acid sequencerelative to another polypeptide sequence. Disclosed variants include,for example, fusion proteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety (such as, for example, polyethylene glycol or albumin,e.g., human serum albumin), phosphorylation, and glycosylation. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Preferably, the anti-EGFR antibodies disclosed herein are characterizedby their variable domain region sequences in the heavy V_(H) and lightV_(L) amino acid sequences. The preferred antibody is A6 which is akappa IgG antibody. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,2nd ed. Raven Press, N.Y. (1989)). The variable regions of eachlight/heavy chain pair form the antibody binding site such that anintact immunoglobulin has two binding sites.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human ErbB3) if it binds to the antigen with a dissociation constant of1 nanomolar or less.

An “antigen binding domain, “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent homology” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

A “host cell” is a cell that can be used to express a nucleic acid. Ahost cell can be a prokaryote, for example, E. coli, or it can be aeukaryote, for example, a single-celled eukaryote (e.g., a yeast orother fungus), a plant cell (e.g., a tobacco or tomato plant cell), ananimal cell (e.g., a human cell, a monkey cell, a hamster cell, a ratcell, a mouse cell, or an insect cell) or a hybridoma. Examples of hostcells include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells(ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivativessuch as Veggie CHO and related cell lines which grow in serum-free media(Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX-B11,which is deficient in DHFR (Urlaub et al., 1980, Proc. Natl. Acad. Sci.USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNAcell line derived from the African green monkey kidney cell line CV1(ATCC CCL 70) (McMahan et al., 1991, EMBO J. 10:2821), human embryonickidney cells such as 293,293 EBNA or MSR 293, human epidermal A431cells, human Colo205 cells, other transformed primate cell lines, normaldiploid cells, cell strains derived from in vitro culture of primarytissue, primary explants, HL-60, U937, HaK or Jurkat cells. Typically, ahost cell is a cultured cell that can be transformed or transfected witha polypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

Antigen Binding Proteins

Antigen binding proteins (e.g., antibodies, antibody fragments, antibodyderivatives, antibody muteins, and antibody variants) are polypeptidesthat bind to EGFR, (preferably, human ErbB3). Antigen binding proteinsinclude antigen binding proteins that inhibit a biological activity ofErbB3.

Oligomers that contain one or more antigen binding proteins may beemployed as ErbB3 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding protein arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple antigenbinding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the antigen binding proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of antigen binding proteins attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourantigen binding proteins. The antigen binding proteins of the oligomermay be in any form, such as any of the forms described above, e.g.,variants or fragments. Preferably, the oligomers comprise antigenbinding proteins that have ErbB3 binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of Fusion Proteins Comprising CertainHeterologous Polypeptides Fused to Various Portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn etal., 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11.

One embodiment is directed to a dimer comprising two fusion proteinscreated by fusing an ErbB3 binding fragment of an anti-ErbB3 antibody tothe Fc region of an antibody. The dimer can be made by, for example,inserting a gene fusion encoding the fusion protein into an appropriateexpression vector, expressing the gene fusion in host cells transformedwith the recombinant expression vector, and allowing the expressedfusion protein to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yield thedimer.

The term “Fc polypeptide” includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization also are included. Fusion proteins comprising Fc moieties(and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al., 1994, FEBS Letters 344:191. The use of amodified leucine zipper that allows for stable trimerization of aheterologous protein fused thereto is described in Fanslow et al., 1994,Semin. Immunol. 6:267-78. In one approach, recombinant fusion proteinscomprising an anti-ErbB3 antibody fragment or derivative fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomeric anti-ErbB3 antibody fragments or derivatives thatform are recovered from the culture supernatant.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab′)₂ fragments.

The present disclosure provides monoclonal antibodies that bind toErbB3. Monoclonal antibodies may be produced using any technique knownin the art, e.g., by immortalizing spleen cells harvested from thetransgenic animal after completion of the immunization schedule. Thespleen cells can be immortalized using any technique known in the art,e.g., by fusing them with myeloma cells to produce hybridomas. Myelomacells for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Examples of suitable cell lines for use in mouse fusionsinclude Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; examples of celllines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and48210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6.

Antigen binding proteins directed against ErbB3 can be used, forexample, in assays to detect the presence of ErbB3 polypeptides, eitherin vitro or in vivo. The antigen binding proteins also may be employedin purifying ErbB3 proteins by immunoaffinity chromatography. Blockingantigen binding proteins can be used in the methods disclosed herein.Such antigen binding proteins that function as ErbB3 antagonists may beemployed in treating any ErbB3-induced condition, including but notlimited to various cancers.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit ErbB3-induced biological activity.Disorders caused or exacerbated (directly or indirectly) by theproteolytic activation of ErbB3, examples of which are provided herein,thus may be treated. In one embodiment, the present invention provides atherapeutic method comprising in vivo administration of an ErbB3blocking antigen binding protein to a mammal in need thereof in anamount effective for reducing an ErbB3-induced biological activity.

Antigen binding proteins include fully human monoclonal antibodies thatinhibit a biological activity of ErbB3.

Antigen binding proteins may be prepared by any of a number ofconventional techniques. For example, they may be purified from cellsthat naturally express them (e.g., an antibody can be purified from ahybridoma that produces it), or produced in recombinant expressionsystems, using any technique known in the art. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Any expression system known in the art can be used to make therecombinant polypeptides of the invention. In general, host cells aretransformed with a recombinant expression vector that comprises DNAencoding a desired polypeptide. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from theAfrican green monkey kidney cell line CV1 (ATCC CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) of ErbB3bound thereto. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian anti-ErbB3 antibodypolypeptides substantially free of contaminating endogenous materials.

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain of thetechniques involve isolating a nucleic acid encoding a polypeptide chain(or portion thereof) of an antigen binding protein of interest (e.g., ananti-ErbB3 antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype (Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover,if an IgG4 is desired, it may also be desired to introduce a pointmutation (CPSCP→CPPCP) in the hinge region (Bloom et al., 1997, ProteinScience 6:407) to alleviate a tendency to form intra-H chain disulfidebonds that can lead to heterogeneity in the IgG4 antibodies.

In particular embodiments, antigen binding proteins of the presentinvention have a binding affinity (K_(a)) for ErbB3 of at least 10⁶. Inother embodiments, the antigen binding proteins exhibit a K_(a) of atleast 10⁷, at least 10⁸, at least 10⁹, or at least 10¹⁰. In anotherembodiment, the antigen binding protein exhibits a K_(a) substantiallythe same as that of an antibody described herein in the Examples.

In another embodiment, the present disclosure provides an antigenbinding protein that has a low dissociation rate from ErbB3. In oneembodiment, the antigen binding protein has a K_(off) of 1×10⁻⁴ to ⁻¹ orlower. In another embodiment, the K_(off) is 5×10⁻⁵ to ⁻¹ or lower. Inanother embodiment, the K_(off) is substantially the same as an antibodydescribed herein. In another embodiment, the antigen binding proteinbinds to ErbB3 with substantially the same K_(off) as an antibodydescribed herein.

In another aspect, the present disclosure provides an antigen bindingprotein that inhibits an activity of ErbB3. In one embodiment, theantigen binding protein has an IC₅₀ of 1000 nM or lower. In anotherembodiment, the IC₅₀ is 100 nM or lower; in another embodiment, the IC₅₀is 10 nM or lower. In another embodiment, the IC₅₀ is substantially thesame as that of an antibody described herein in the Examples. In anotherembodiment, the antigen binding protein inhibits an activity of ErbB3with substantially the same IC₅₀ as an antibody described herein.

In another aspect, the present disclosure provides an antigen bindingprotein that binds to human ErbB3 expressed on the surface of a celland, when so bound, inhibits ErbB3 signaling activity in the cellwithout causing a significant reduction in the amount of ErbB3 on thesurface of the cell. Any method for determining or estimating the amountof ErbB3 on the surface and/or in the interior of the cell can be used.In other embodiments, binding of the antigen binding protein to theErbB3-expressing cell causes less than about 75%, 50%, 40%, 30%, 20%,15%, 10%, 5%, 1%, or 0.1% of the cell-surface ErbB3 to be internalized.

In another aspect, the present disclosure provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO00/09560, incorporated by reference herein.

The present disclosure further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of ErbB3, or to anepitope of ErbB3 and an epitope of another molecule, via two differentantigen binding sites or regions. Moreover, bispecific antigen bindingprotein as disclosed herein can comprise an ErbB3 binding site from oneof the herein-described antibodies and a second ErbB3 binding regionfrom another of the herein-described antibodies, including thosedescribed herein by reference to other publications. Alternatively, abispecific antigen binding protein may comprise an antigen binding sitefrom one of the herein described antibodies and a second antigen bindingsite from another ErbB3 antibody that is known in the art, or from anantibody that is prepared by known methods or the methods describedherein.

Numerous methods of preparing bispecific antibodies are known in theart. Such methods include the use of hybrid-hybridomas as described byMilstein et al., 1983, Nature 305:537, and chemical coupling of antibodyfragments (Brennan et al., 1985, Science 229:81; Glennie et al., 1987,J. Immunol. 139:2367; U.S. Pat. No. 6,010,902). Moreover, bispecificantibodies can be produced via recombinant means, for example by usingleucine zipper moieties (i.e., from the Fos and Jun proteins, whichpreferentially form heterodimers; Kostelny et al., 1992, J. Immunol.148:1547) or other lock and key interactive domain structures asdescribed in U.S. Pat. No. 5,582,996. Additional useful techniquesinclude those described in U.S. Pat. Nos. 5,959,083; and 5,807,706.

In another aspect, the antigen binding protein comprises a derivative ofan antibody. The derivatized antibody can comprise any molecule orsubstance that imparts a desired property to the antibody, such asincreased half-life in a particular use. The derivatized antibody cancomprise, for example, a detectable (or labeling) moiety (e.g., aradioactive, colorimetric, antigenic or enzymatic molecule, a detectablebead (such as a magnetic or electrodense (e.g., gold bead), or amolecule that binds to another molecule (e.g., biotin or streptavidin),a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, orpharmaceutically active moiety), or a molecule that increases thesuitability of the antibody for a particular use (e.g., administrationto a subject, such as a human subject, or other in vivo or in vitrouses). Examples of molecules that can be used to derivatize an antibodyinclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antibodies can beprepared using techniques well known in the art. In one embodiment, theantibody is conjugated or otherwise linked to transthyretin (TTR) or aTTR variant. The TTR or TTR variant can be chemically modified with, forexample, a chemical selected from the group consisting of dextran,poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycolhomopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols and polyvinyl alcohols.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

Indications

In one aspect, the present disclosure provides methods of treating asubject. The method can, for example, have a generally salubrious effecton the subject, e.g., it can increase the subject's expected longevity.Alternatively, the method can, for example, treat, prevent, cure,relieve, or ameliorate (“treat”) a disease, disorder, condition, orillness (“a condition”). Among the conditions to be treated areconditions characterized by inappropriate expression or activity ofErbB3. In some such conditions, the expression or activity level is toohigh, and the treatment comprises administering an ErbB3 antagonist asdescribed herein. The disorders or conditions are cancer-related. Inparticular, those cancers include, but are not limited to, breast, lung,ovarian and colon carcinoma and various myelomas.

Specific medical conditions and diseases that are treatable orpreventable with the antigen binding proteins of this disclosure includevarious cancers.

Therapeutic Methods and Administration of Antigen Binding Proteins

Certain methods provided herein comprise administering an ErbB3 bindingantigen binding protein to a subject, thereby reducing an ErbB3-inducedbiological response that plays a role in a particular condition. Inparticular embodiments, methods of the invention involve contactingendogenous ErbB3 with an ErbB3 binding antigen binding protein, e.g.,via administration to a subject or in an ex vivo procedure.

The term “treatment” encompasses alleviation or prevention of at leastone symptom or other aspect of a disorder, or reduction of diseaseseverity, and the like. An antigen binding protein need not effect acomplete cure, or eradicate every symptom or manifestation of a disease,to constitute a viable therapeutic agent. As is recognized in thepertinent field, drugs employed as therapeutic agents may reduce theseverity of a given disease state, but need not abolish everymanifestation of the disease to be regarded as useful therapeuticagents. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur or worsen in a subject, is sufficient. Oneembodiment of the invention is directed to a method comprisingadministering to a patient an ErbB3 antagonist in an amount and for atime sufficient to induce a sustained improvement over baseline of anindicator that reflects the severity of the particular disorder.

As is understood in the pertinent field, pharmaceutical compositionscomprising the antibodies and fragments thereof of the disclosure areadministered to a subject in a manner appropriate to the indication.Pharmaceutical compositions may be administered by any suitabletechnique, including but not limited to, parenterally, topically, or byinhalation. If injected, the pharmaceutical composition can beadministered, for example, via intra-articular, intravenous,intramuscular, intralesional, intraperitoneal or subcutaneous routes, bybolus injection, or continuous infusion. Localized administration, e.g.at a site of disease or injury is contemplated, as are transdermaldelivery and sustained release from implants. Delivery by inhalationincludes, for example, nasal or oral inhalation, use of a nebulizer,inhalation of the antagonist in aerosol form, and the like. Otheralternatives include eyedrops; oral preparations including pills,syrups, lozenges or chewing gum; and topical preparations such aslotions, gels, sprays, and ointments.

Use of antigen binding proteins in ex vivo procedures also iscontemplated. For example, a patient's blood or other bodily fluid maybe contacted with an antigen binding protein that binds ErbB3 ex vivo.The antigen binding protein may be bound to a suitable insoluble matrixor solid support material.

Advantageously, antigen binding proteins are administered in the form ofa composition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Optionally,the composition additionally comprises one or more physiologicallyactive agents, for example, a second inflammation- or immune-inhibitingsubstance, an anti-angiogenic substance, an analgesic substance, etc.,non-exclusive examples of which are provided herein. In variousparticular embodiments, the composition comprises one, two, three, four,five, or six physiologically active agents in addition to an EGFRbinding antigen binding protein

Combination Therapy

In another aspect, the present disclosure provides a method of treatinga subject with an ErbB3 inhibiting antigen binding protein and one ormore other treatments. In one embodiment, such a combination therapyachieves synergy or an additive effect by, for example, attackingmultiple sites or molecular targets in a tumor. Types of combinationtherapies that can be used in connection with the present inventioninclude inhibiting or activating (as appropriate) multiple nodes in asingle disease-related pathway, multiple pathways in a target cell, andmultiple cell types within a target tissue.

In a preferred embodiment, an anti-ErbB3 antibody provided herein isadministered together with an antibody selected from the groupconsisting of an anti-HER2 antibody, an anti-HER3 antibody, an anti-EGFRantibody and combinations thereof (see examples 10 and 11 below).

In another embodiment, a combination therapy method comprisesadministering to the subject two, three, four, five, six, or more of theErbB3 agonists or antagonists described herein. In another embodiment,the method comprises administering to the subject two or more treatmentsthat together inhibit or activate (directly or indirectly)ErbB3-mediated signal transduction. Examples of such methods includeusing combinations of two or more ErbB3 inhibiting antigen bindingproteins, of an ErbB3 inhibiting antigen binding protein and one or moreother therapeutic moiety having anti-cancer properties (for example,cytotoxic agents, and/or immunomodulators), or of an ErbB3 inhibitingantigen binding protein and one or more other treatments (e.g., surgery,or radiation). Furthermore, one or more anti-ErbB3 antibodies orantibody derivatives can be used in combination with one or moremolecules or other treatments, wherein the other molecule(s) and/ortreatment(s) do not directly bind to or affect ErbB3, but whichcombination is effective for treating or preventing the condition beingtreated. In one embodiment, one or more of the molecule(s) and/ortreatment(s) treats or prevents a condition that is caused by one ormore of the other molecule(s) or treatment(s) in the course of therapy,e.g., nausea, fatigue, alopecia, cachexia, insomnia, etc. In every casewhere a combination of molecules and/or other treatments is used, theindividual molecule(s) and/or treatment(s) can be administered in anyorder, over any length of time, which is effective, e.g.,simultaneously, consecutively, or alternately. In one embodiment, themethod of treatment comprises completing a first course of treatmentwith one molecule or other treatment before beginning a second course oftreatment. The length of time between the end of the first course oftreatment and beginning of the second course of treatment can be anylength of time that allows the total course of therapy to be effective,e.g., seconds, minutes, hours, days, weeks, months, or even years.

In another embodiment, the method comprises administering one or more ofthe ErbB3 antagonists described herein and one or more other treatments(e.g., a therapeutic or palliative treatment). Where a method comprisesadministering more than one treatment to a subject, it is to beunderstood that the order, timing, number, concentration, and volume ofthe administrations is limited only by the medical requirements andlimitations of the treatment, i.e., two treatments can be administeredto the subject, e.g., simultaneously, consecutively, alternately, oraccording to any other regimen.

Example 1

This example describes in vitro data showing the binding specificity ofanti-ErbB3 antibodies, relative to other members of the ErbB (EGFR)family. The binding of the antibodies towards mouse ErbB3 was alsoassessed to check for the cross reactivity towards the mouse antigen ofinterest.

Costar Clear EIA/RIA 96-Well, half area plates were coated withappropriated human recombinant proteins (ErbB3, EGFR, or ERbB2) or mouseErbB3 and incubated overnight at 4° C.

Plates were washed three times with PBS. Wells were blocked for 1H atroom temperature with Casein in PBS. Plates were washed three times withPBS. Anti-ErbB3 antibodies or phage soup were added to wells andincubated for 1 hour at room temperature. Plates were washed three timeswith PBS prior to addition of appropriate peroxidase-linked detectionantibody for one hour at room temperature. After three washes, TMBsubstrate (Pierce), followed by stop solution (2 M H₂SO₄) were added asrecommended in manufacturer's protocol. The binding of ErbB3 antibodieswas determined by measuring the OD450 using a SpectraMax plate readerand analyzed using the Microsoft EXCEL program.

Results are depicted in FIG. 1A and show that anti-ErbB3 antibodiesspecifically bind to recombinant ErbB3 in an ELISA, but do not show anyappreciable binding to EGFR, or ErbB2. FIG. 1B shows that anti-ErbB3antibodies cross-react with mouse ErbB3.

Example 2

This example illustrates the binding of anti-ErbB3 antibodies toendogenous human ErbB3 expressed on human cancer cells, as assayed byflow cytometry. EC₅₀ values for antibodies were determined as follows.

Her3 expressing MCF7 cells were harvested with enzyme-free CellDissociation Buffer (GIBCO) and transferred to V-Bottom 96 well-plates(50,000 cells/well). Cells were incubated on ice for 30 min with serialdilutions of anti-ErbB3 antibodies in FACS buffer (PBS+2% FBS)+NaN₃.After 2 washes in FACS buffer, a 1:1000 dilution of Phycoerythrinconjugated anti-Human IgG (γ-chain specific) was added and incubated for30 min. Following a final wash, fluorescence intensity was measured onan Intellicyt High Throughput Flow Cytometer (HTFC). Data were analyzedusing Graphpad Prism software using non-linear regression fit. Datapoints are shown as the median fluorescence intensity (MFI) ofpositively labeled cells +/− Standard Error. EC₅₀ values are reported asthe concentration of antibody to achieve 50% of maximal ErbB3 antibodiesbinding to ErbB3 expressing cells. In parallel, cell binding was alsoassessed with the same method on CHO—K1 cells, which do not expresshuman ErbB3 endogenously and thus served as a negative control.

As shown in FIG. 2 and Table 1, selected antibodies strongly bound ErbB3expressing MCF7 cells, and displayed EC₅₀ values ranging from 0.049 to5.97 nM. Most of the antibody showed little or no binding to cells notexpressing human ErbB3 (CHO-K1 cells). These data altogether demonstratea strong and specific binding of the antibodies to endogenous ErbB3.

Table 1 shows binding characteristics (EC₅₀, in Mol.L⁻¹) of anti-ErbB3antibodies to cells either expressing ErbB3 (MCF7) or not (CHO-K1). Theresults indicate that most anti-ErbB3 antibodies bind specifically toErbB3-positive Cells.

TABLE 1 ErbB3 (+) Cell Line ErbB3 (−) Cell Line EC50 (M) (MCF7) (CHO)A01 5.71E−09 — A02 1.222E−09 3.519E−08 A04 1.628E−10 — A08 7.472E−10 —A11 2.19E−10 — B01 2.647E−09 — B08 9.215E−11 — B11 4.902E−11 — C034.266E−10 2.16E−09 D01 4.025E−09 >1.0E−06 D03 5.972E−09 — D04 2.421E−11— D07 9.332E−10 — D08 1.933E−09 — D10 2.01E−09 — E01 2.53E−09 1.305E−08E08 3.067E−09 >1.0E−08

Example 3

This example illustrates Analytical Size-Exclusion Chromatography(ANSEC) analysis of anti-ErbB3 monoclonal antibodies was performed undernative conditions on a Waters Breeze HPLC system coupled to an externalWyatt Technology miniDAWN Treos multi-angle light scattering (MALS)system. Chromatography separation was performed in PBS pH 7.4 buffer at0.5 mL/min flow rate using BioSep-SEC-s3000, 300×7.8 mm column(Phenomenex). The duration of each run was 30 minutes. Prior to processsamples, the column running conditions were confirmed with BIO-RAD gelfiltration protein standard (Cat #, 151-1901: Thyroglobin=670 KDa;Gamma-globulin=158 KDa; Ovalbumin=44 KDa; Myoglobin=17 KDa; VitaminB12=1.35 KDa). Absolute molecular weight of single homogeneous proteinband separated on ANSEC run was evaluated using online MALS system with≧95% confidence.

The results are shown in FIG. 3 which provide overlaid ANSECchromatograms (Ultra Violet trace at 280 nm) of selected anti-ErbB3antibodies as well as Bio-Rad Standard (grey square dot) under isocraticelution conditions. All antibodies displayed homogenous distribution,with no detectable aggregation observed. Table 2 indicates the averagemolecular weights determined for selected antibodies.

Example 4

This example illustrates in vitro data showing the inhibition ofHeregulin-stimulated cell proliferation by anti-ERbB3 antibodies.

In this example, 10,000 MCF7 breast cancer cells were plated into eachwell of a 96-well cell culture plate in complete culture media. After 24hr, media was removed, and cells were incubated with variousconcentrations of antibodies in DMEM+0.5% FBS for 45 min, followed byaddition of NRG1-beta 1 to a final concentration of 10 ng/ml. Cells wereincubated for 72 hr at 37° C., after which the Promega Cell Titer 96Non-radioactive Cell Proliferation (MTT) Assay kit was used to evaluatecell proliferation, as recommended by manufacturer's protocol. Theproliferative index was calculated as the OD₅₇₀ of NRG1-beta 1 treatedsample (with or without antibody treatment) relative to untreated cells.Data were analyzed using Graphpad Prism software using non-linearregression fit.

Results, shown in FIG. 4, indicate that anti-ErbB3 antibodies inhibitcell proliferation induced by NRG1-beta 1. Among them, A01, A02, and A08were the most potent, with IC₅₀ values of 10.6 nM, 2.8 nM and 12.8 nMrespectively.

Table 2 indicates the estimated molecular weights for the selectedantibodies, as determined by ANSEC-MALS.

TABLE 2 MW on MALS Sample ID (+−5kDa) ErbB3- A01 172.5 ErbB3- A02 146.3ErbB3- A04 140 ErbB3- A08 149.9 ErbB3- A11 162.5

Example 5

This example illustrates in vitro data showing NRG1 beta-1 stimulatedphosphorylation of Her3 in MCF7 breast cancer cells. This exampledemonstrates the ability of antibodies to block the activation of ErbB3,in cancer cells, and therefore its function potentially. Typically,20,000 MCF7 cells were plated in the wells of a 96-well cell culturecluster in 100 μl Phenol Red-free DMEM media supplemented with 10% FBS.Cells were then starved for 20 hr in 100 μl starvation media (PhenolRed-free DMEM+0% FBS). Antibodies were diluted to 20 μg/ml (2× finalconcentration) in 50 μl serum-free media, then added to the cells afterremoval of starvation media. After 3 hr incubation, 50 μl of 20 ng/mlNRG1-beta1 was added to the cells for a final concentration of 10 ng/ml.After 10 min, cells were washed with ice-cold PBS supplemented withNaVO₄, and then lyzed in 1× Cell Lysis Buffer (Cell SignalingTechnology). Phosphorylation of Her3 was detected using PathScan®Phospho-HER3/ErbB3 (panTyr) Sandwich ELISA Kit according tomanufacturer's protocol (Cell Signaling Technology) adjusted for halfarea ELISA plates. The effect of antibody was determined by measuringthe OD450 using a SpectraMax plate reader and analyzed using GraphPadPrism 5 software.

The results, shown in FIG. 5, indicate that anti-ErbB3 A01, A02, and A04antibodies strongly inhibit ErbB3 phosphorylation induced by Heregulin,in a dose dependent manner, with IC50 values of 0.46, 0.12, and 0.13 nMrespectively.

Example 6

This example illustrates in vitro data showing NRG1 beta-1 stimulatedphosphorylation of ERK1/2 MAPK in MCF7 breast cancer cells. This exampledemonstrates the ability of antibodies to block the activation of ERK1/2and hereby the function of Her3 in cancer cells. Typically, 20,000 MCF7cells were plated in the wells of a 96-well cell culture cluster in 100μl Phenol Red-free DMEM media supplemented with 10% FBS. Cells were thenstarved for 20 hr in 100 starvation media (Phenol Red-free DMEM+0% FBS).Antibodies were diluted to 20 μg/ml (2× final concentration) in 50 μlserum-free media, and added to the cells after removal of starvationmedia. After 3 hr incubation, 50 μl of 20 ng/ml NRG1 beta 1 was added tothe cells for a final concentration of 10 ng/ml. After 10 min, cellswere washed with ice-cold PBS supplemented with NaVO₄, and then lyzed in1× Cell Lysis Buffer (Cell Signaling Technology). Phosphorylation ofHer3 was detected using PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204)Sandwich ELISA Antibody Pair according to manufacturer's protocol (CellSignaling Technology) adjusted for half area ELISA plates. The effect ofantibody was determined by measuring the OD450 using a SpectraMax platereader and analyzed using GraphPad Prism 5 software.

The results, shown in FIG. 6, indicate that anti-ErbB3 A01, A02, and A04antibodies strongly inhibit ERK1/2 phosphorylation induced by NRG1beta-1, in a dose dependent manner, with IC50 values of 0.46, 0.12, and0.13 nM respectively.

Example 7

This example illustrates the ability of antibodies to block theinteraction between Her3 receptor and its natural ligand NRG1-beta 1(Heregulin/Neuregulin). Recombinant human NRG1-b1 was immobilized onAR2G sensor using standard NHS/EDC coupling methodology. Thenrecombinant human ErbB3/Fc was loaded alone (2 μg/ml final) orpre-incubated with anti-ErbB3 antibodies, in mixtures of 2 μg/ml and 20μg/ml final respectively. The binding of ErbB3 in the presence of theantibodies was assessed on a ForteBio's Octet® platform. As shown inFIG. 7, results indicate that A01 and A08 strongly inhibited the bindingof ErbB3 to its ligand, whereas A04 and A02 only partially inhibited thebinding.

Example 8

This example illustrates the ability of anti-ErbB3 antibodies to inducereceptor internalization, a prerequisite for a decrease in ErbB3 cellsurface expression, and thus for the inhibition of ErbB3 function incancer cells.

Cells were plated at 20.000 cells in each well of 96 well plates. Theday after, cells were starved in serum-free media for 20H. Media wasthen replaced with serum free media containing 10 ug/ml of anti-ErbB3antibody, or a non relevant control antibody, or no antibody, forincreasing periods of time, ranging from 0 to 90 min. Cells were thenwashed briefly once with trypsin, then three times with PBS, and fixedin paraformaldehyde (4% in PBS) for 10 min at room temperature. After 3washes in PBS, cells were permeabilised with Blocker Casein in PBS(Pierce)+0.25% NP40 for 10 min at room temperature, washed 3 times againand incubated with Blocker Casein in PBS for 30 min. Anti-humanErbB3-Phycoerythrin was then added to 1/1000 final dilution and cellswere further incubated at room temperature for 1H. Following 3 washes,cell nuclei were labeled with Hoechst for 5 min. Receptor localizationwas assessed on an EVOS-Fl microscope and images were analyzed withImageJ software.

Results, shown in FIG. 8, illustrate the levels of ErbB3 on surfaces ofMCF7 cells in the absence and the presence of an exemplary anti-ErbB3antibody. After 1 hour incubation with A04, ErbB3 is mainly detected inthe cytoplasmic/endosomal compartment of the cells, whereas in thepresence of the control antibody (data not shown), or in the absence ofantibody, ErbB3 is mostly detected at the cell surface.

Example 9

This example illustrates the ability of anti-ErbB3 antibodies to inhibitHer3 dependent cell migration. Migration assays were set up using CIM-16plates with 8 μm pore membranes according to manufacturer's protocol(Roche). Wells were coated with 30 μl gelatin 0.1% in H₂O (EMDMillipore) on each side. Meanwhile, MCF7 cells were starved inserum-free media for 4H. Then the wells of the bottom chambers werefilled with 160 μL of serum-free media supplemented or not with 10%serum. The top and bottom portions of the CIM-16 plates were assembledtogether, and the assembled CIM-16 plate was allowed to equilibrate for1 hour at 37° C. after the addition of 30 μL of serum-free media to thetop chamber wells. Cells were harvested with cell dissociation buffer,resuspended in serum-free media and 40,000 cells were added to eachwell, in presence or not of 10 μg/ml antibody. After incubation at roomtemperature for 30 min, CIP-16 plates were placed into the xCELLigenceRTCA MP system and data collection started. Impedance values (reportedas cell index values) were collected every 15 min for 20 hours. Theslopes of the curves thus generated for each condition were plottedusing the xCELLigence and EXCEL softwares. Results, shown in FIG. 9,illustrate that an exemplary antibody, A04, efficiently inhibited MCF7migration.

Example 10

This example illustrates in vitro data showing the inhibition of FBS-and Heregulin-stimulated cell proliferation by a combination ofanti-ErbB3 and anti-EGFR antibodies. In this example, 2,000 A431NSadenocarcinoma cells were plated into each well of a 96-well cellculture plate in complete culture media. After 24 hr, media was removed,and cells were incubated with various concentrations of antibodies inDMEM+0.5% FBS for 45 min, followed by addition of NRG1-beta 1 to a finalconcentration of 25 ng/ml. Cells were incubated for 72 hr at 37° C.,after which the Promega Cell Titer 96 Non-radioactive Cell Proliferation(MTT) Assay kit was used to evaluate cell proliferation, as recommendedby manufacturer's protocol. Proliferation was expressed in RelativeLuminescence Units (RLU) and data were analyzed using Graphpad Prismsoftware using non-linear regression fit.

Results are shown in FIG. 10. A431NS cells are carcinoma cell lineaggressively proliferation under [0.5% FBS+NRGB1] stimulation.Individually, anti-EGFR (A06 or Erbitux) and anti-Her3 IgGs (A02) haveonly a modest inhibitory effect on A431NS proliferation. However whencombined, anti-EGFR and anti-Her3 IgG show robust dose-dependentinhibition of cell proliferation.

Example 11

This example illustrates in vitro data showing the inhibition of FBS-and/or Heregulin-mediated activation of the ErbB family pathways, by acombination of anti-ErbB3 antibody with either anti-EGFR or anti-Her2antibodies.

Approximately, 20,000 A431NS cells were plated in the wells of a 96-wellcell culture cluster in 100 μl Phenol Red-free DMEM media supplementedwith 10% FBS. Cells were then starved for 20 hr in 100 μl starvationmedia (Phenol Red-free DMEM+0% FBS). Antibodies were diluted to 20 μg/ml(2× final concentration) in 50 μl serum-free media, then added to thecells after removal of starvation media. After 1 hr incubation, 50 μl ofNRG1-beta 1 (5 ng/ml final) or FBS (0.5% final) or both, were added tothe cells. After 10 min at 37° C., cells were washed with ice-cold PBSsupplemented with NaVO₄, and then lyzed in 100 μl of Cell Lysis Buffer(Cell Signaling Technology). Phosphorylation of Akt and MAPK wasdetected using PathScan® Sandwich ELISA Kits Phospho-Akt1 andphospho-p44/42 MAPK (Cell Signaling Technology #7147 and #7246respectively) according to manufacturer's protocol, adjusted for halfarea ELISA plates. The effect of antibody was determined by measuringthe OD₄₅₀ using a SpectraMax plate reader and analyzed using GraphPadPrism 5 software.

The results are shown in FIG. 11. NRG-beta-1 mostly activates ErbB3receptor, whereas FBS, which contains EGF but not NRG-beta-1, is believeto stimulate EGFR predominantly. Her2 receptor does not have any knownligand, but is known to interact with both EGFR and ErbB3 upon theiractivation. Data in FIG. 11 show that individually, anti-ErbB3 A02,anti-EGFR Cetuximab, and anti-Her2 Trastuzumab, all showed relativeinhibition of both MAPk and Akt phosphorylation. Most importantly,synergetic effect was observed when combined in pairs. For example,robust inhibition of MAPK phosphorylation was observed when anti-ErbB3antibodies A02 and Cetuximab were combined, whereas combination ofanti-ErbB3 and anti-Her2 Trastuzumab predominantly inhibited Aktphosphorylation. These data illustrate the potential in targetingdifferent receptors involved in the same signaling cascade.

Sequence Listing Heavy chain variable domain regionLight chain variable domain region A01QVQLVQSGAEVKQPGASVKVSCKASGGTFSSYAIS QSALTQPPSASGSPGQSVTISCTGTSSDVGAWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVT YNDVSWYQHHPGKAPKLIIYEVNHRPSGVPITADESTSTAYMELSSLRSEDTAVYYCATTTMVRGVI DRFSGSKSDNTASLTISGLQAEDEADYYCSSMYYFDYWGQGTLVTVSS SEQ ID NO. 1 YTPSNTPLVFGSGTKVTVL SEQ ID NO. 2 A02EVQLVQSGAEVKKPGASVTVSCKPSGYAFTDYYIHW QPVLTQPPSVSAAPGQRVTISCSGSSSNIGNVRQAAGQGLEWMGWVDPRSGATVYAQNFQGRV NYLSWYQQLPGTAPKLLIYDDTKRPSGIPDRTMTRDRSSSTVYMDLSRVTSDDTALYFCGRDNYGIF FSGSKSGTSATLGITGLQTGDEADYYCGTWDYWGQGTLVTVSS SEQ ID NO. 3 DGSLDAEVFGTGTKLTVL SEQ ID NO. 4 A04QVQLQQSGPGLVQPSQTLSLTCDISGDSVSSTGAA QTVVTQAPSVSVAPGQTARITCGGNNIGSKWNWIRQSPSRGLEWLGRTYYRSKWYNDYALSVRS SVHWYQQKPGQAPVLVVYDDSDRPSGIPERITITPDTSKNQFSLQLNSVTPEDTAVYYCVRDGDVG RFSGSNSGNTATLTISRVEAGDEADYYCQVLDAFDIWGQGTMVTVSS SEQ ID NO. 5 WDSTSDHVVFGGGTKVTVL SEQ ID NO. 6 A08QMQLVQSGADVKKPGASVRVSCKTSGYTFTSYDIN QSVLTQPPSVSVAPGNTATITCGGDNIGEKTWVRQAPGQGLEWMGWMNPDTGNTAYVQKFQG VHWYQQRPGQAPALVIYYDSDRPSGIPERFRVTVTRDTSINTVYMELSGLRSEDTAVYYCAKSGGSS SGSNSGNTATLTISRVEAGDEADYYCQVWDSYDAFDIWGQGTMVTVSS SEQ ID NO. 7 SSSDHVVFGGGTKLTVL SEQ ID NO. 8 A11QVQLVQSGRGLVQPGRSLRLSCAASGFTFDEYAMH EIVLTQSPSSLSASVGDRVTITCQASHDISNYWVRHAPGKGLEWVAGMSWNGGGIGYADSVKGR LTWYQHRPGRAPKLLIFDASRLETGVPSRFSFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGAGG GYGSGKDFTFTISGLQPEDFATYYCQHYADLSYPTDAFDIWGQGTTVTVSS SEQ ID NO. 9 PSFGQGTKLEIK SEQ ID NO. 10 A12EVQLLESGAEVKKPGASMKVSCKASGYPFPSYGISW QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVRQAPGQGLEWMGWINPYNGETNSAQQLQGRV VHWYQQKPGQAPVLVIYYDSDRPSGIPERFTMTTDTSASTAYMELRSLRADDTAVYYCARARTPIA SGSNSGNTATLTISRVEAGDEADYYCQVWDAAWFDPWGQGTLVTVSS SEQ ID NO. 11 SSSDHLVFGTGTKLVT SEQ ID NO. 12 B01QVQLVESGGGLVQPGGSLRLACAASGFTFSNYEMN QAGLTQPPSASASPGDSVTLTCTLSSDHSDYWVRQAPGKGLEWVATISDDSVYKYYADSVKGRFTI KVDWYQQRPGKGPRFVMRVGTGGIVGVKSRDNSKALFYLQMDSLTFEDTAIYYCARDPGGDSLG VSNRFSGSKSGNTASLTISGLQAEDEADYYCYFQHWGQGTLVTVSS SEQ ID NO. 13 SSYTDSSTLDVVFGGGTKLTVL SEQ ID NO. 14 B03EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYSMN SYELMQPPSVSVAPGKTARITCGGNNIGSKSWVRQAPGKGLEWVSSISSSSSYIFYADSVKGRFTISR VHWYQQKPGQAPVLVIYYDSDRPSGIPERFDNAKNSLYLQVNSLGAEDTAVYYCARASSEDAFDI SGSNSGNTATLTISRVEAGDEADYYCQVWDWGQGTMVTVSS SEQ ID NO. 15 SSSDHVVFGGGTKVTVL SEQ ID NO. 16 B04QLVESGGGVVQPGGSLRLSCAASGFNFRAYGMHW QAGLTQPPSVSVAPGKTARITCGGDNIGSKSVRQAPGKGLEWLAMMSSDGTRTYYADSVKGRFTIS VHWYQQKPGQAPVLVIYYDTDRPSGIPERFRDNSKNTLYVQMNSLRAEDTAVYYCARDRGSFSSS SGSNSGNTATLTISRVEAGDEADYYCQVWDFDLWGQGTLVTVSS SEQ ID NO. 17 SSSDPVVFGGGTKLTVL SEQ ID NO. 18 B05EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYWMT QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAWVRQAPGKGLEWVASMNQDGSEKYYVDSVKGRF GYEVHWYQHVPGTAPKLLIYINSNRPSGIPNTISRDNAKNSLYLQMNSLRDEDTALYYCAKAYSSSW RFSGSKSGNTAFLTISGLQAEDEADYYCISYTLLSGGMDVWGQGTTVTVSS SEQ ID NO. 19 SSNTWVFGGXTKVTVL SEQ ID NO. 20 B08QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHYGIS QSVLTQPPSASGSPGQSVTISCTGTSSDVGGWVRQAPGQGLEWMGSITPHNGKTNYVQMFQGR YNFVSWYQQHPGKAPKLMIYDVTNRPSGVVTMTTDTSTRTVYMELRNLRSDDTAVYYCARDWN SNRFSGSKSGNTASLTISGLQAEDEADYYCSFADFDYWGQGTLVTVSS SEQ ID NO 21 SYTSSSTPYVFGTGTKLTVL SEQ ID NO. 22 B10QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMN QPVLTQPPSVSVAPGKTARITCGGNNIGSKSWVRQAPGKGLEWVSSISSRSGYIYYADSVKGRFTISR VHWYQQKPGQAPVLVIYYDSDRPSGIPERFDNAKNSLYLQMNSLRDEDTAVYYCVRDGGTVDAF SGSNSGNTATLTISRVEAGDEADYYCQVWDDIWGQGTMVTVSS SEQ ID NO 23 SSSDHVVFGGGTKLTVL SEQ ID NO. 24 B11QVQLVQSGRGLVQPGRSLRLSCAASGFTFDEYAMH EIVLTQSPSSLSASVGDRVTITCQASHDISNYWVRHAPGKGLEWVAGMSWNGGGIGYADSVKGR LTWYQHRPGRAPKLLIFDASRLETGVPSRFSFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGAGG GYGSGKDFTFTISGLQPEDFATYYCQHYADLSYPTDAFDIWGQGTTVTVSS SEQ ID NO 25 PSFGQGTKVDIK SEQ ID NO. 26 C03QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISW SYELMQPPSVSVAPGKTARITCGGNNIGSKSVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVT VHWYQQKPGQAPVLVIYYDSDRPSGIPERFMTTDTSTSTAYMELRSLRSDDTAVYYCAREAYAWG SGSNSGNTATLTISRVEAGDEADYYCQVWDAFDIWGQGTMVTVSS SEQ ID NO 27 SSRDHAVFGGGTKLTVL SEQ ID NO. 28 C07QVQLQQSGPGLVQPSQTLSLTCDISGDSVSSTGAA QAVVTQPPSVSVAPGQTARITCAGNNIGSKWNWIRQSPSRGLEWLGRTYYRSKWYNDYALSVRS SVHWYQQKPGQAPVLVIFDDSDRPSGISERRITITPDTSKNQFSLQLNSVTPEDTAVYYCVRDGDVG FSGSNSGNTATLTISRVEAGDEADYYCQVWLDAFDIWGQGTMVTVSS SEQ ID NO 29 DGDTDHVVFGGGTKLTVL SEQ ID NO. 30 C10QVQLQQWGAGLLKSSETLSLSCAVYGGTFRDDHW QPVLTQPASVSGSPGQSITISCTGTSSDVGGSWIRQPPGKGLEWIGESHHTGRTIYNPSLRSRVTMS YNYVSWYQQHPGKAPKLMIYDVSKRPSGVIDTSKNEFSLILRSVTAADTATYFCARGNNYVWGNQ SNRFSGSKSGNTASLTISGLQAEDEADYYCSEDFWGQGTLVTVSS SEQ ID NO 31 SYTSNSIYVFGTGTKVTVLG SEQ ID NO. 32 C12QLVQSGAEVRKPGASVKVSCKASGYTFADYYIHWV QSVVTQPPSVSAAPGQKVTISCSGSSSNIGNRQAPGQGLEWMGWINPNSGGADYAQKLQGRVT NYVSWYQQLPGTAPKLLIYDNNERPSGIPDMTTDTSTSTAYMELRSLRSDDTAVYYCAREGYAWG RFSGSKSGTSATLGITGLQTGDEADYYCGTAFDIWGQGTMVTVSS SEQ ID NO 33 WDSSLSAGVFGGGTKLTVL SEQ ID NO. 34 D01EVQLVQSGAEVRKPGASVKVSCKASQYTFTNYHIH QSVLTQPPSASGTPGQSVTISCSGSSSNIGGWVRQVPGQGLEWMAMINPSNGNTNYGQRFQDR NTVNWYQQFPGTAPKLLMYINNLRPAGVPVTLTRDTSTSTAYMELSSLRSEDTAVYLCARDGLTFG DRFSGSKSGTSASLVISGLQSEDEADYYCASDLLAYWGQGTLVTVSS SEQ ID NO 35 WDDSLSGLVFGGGTKLTVL SEQ ID NO. 36 D03QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMN SYELMQPPSVSVAPGKTARITCGGNNIGSKSWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISR VHWYQQKPGQAPVVVIYYDSDRPSGIPERFDNSKNTLYLQMNSLRDEDTAVYYCAREILTGYYPDA SGSNSGNTATLTISRVEAGDEADYYCQVWDFDMWGQGTMVTVSS SEQ ID NO 37 SSSDHYVFGTGTKLTVL SEQ ID NO. 38 D04EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMNW QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRD VHWYQQKPGQAPLLVIYYDTDRPSGIPERFNAKNSLYLQMNSLRAEDTAVYYCARGFGEDLFDYW SGSNSENTATLTISGVEAGDEADYYCQVWDGQGTLVTVSS SEQ ID NO 39 IGSDQVVFGTGTKVTVL SEQ ID NO. 40 D07QVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMS SYELTQPASVSGSPGQSITISCTGTSSDVGNYWVRQAPGKGLEWVAYINLDGSEKDYVDSVKGRFT! NSVSWYQQHPGKAPELMIYDVNNRPSGVSSRDNAKNSLYLQMNSLRAEDTALYYCAKAYSSSWLL NRFSGSKSGNTASLTISGLQAEDEADYYCSSSGGMDVWGQGTTVTVSS SEQ ID NO 41 YTSSSTLVFGGGTQLTVLG SEQ ID NO. 42 D08QVQLVESGGGLVKPGGSLRLSCAASGFTSSSYVLNW QSVLTQPASVSGSPGQSITISCTGSSSDVGSYVRQAPGKGLEWVSSISSRGSYIYYADSVKGRFTISRD NLVSWYQQHPGKAPKLMIYDDNRRPSGISTNAKNSLYLQMNSLRAEDTAVYYCARERAGFSPVNA RFSGSKSGNTASLTVSGLQAEDEAYYYCSSYFDIWGQGTMVTVSS SEQ ID NO 43 AGRNNDVIFGGGTQLTVL SEQ ID NO. 44 D10QVQLVESGGGLVQPGGSLRLACAASGFTFSNYEMN QAGLTQPPSASASPGDSVTLTCTLSSDHSDYWVRQAPGKGLEWVATISDDSVYKYYADSVKGRFT! KVDWYQQRPGKGPRFVMRVGTGGIVGVKSRDNSKALFYLQMDSLTFEDTAIYYCARDPGGDSLG GDGIPDRFSVWGSGPNRYLTIKNIQEEDESDYFQHWGQGTLVTVSS SEQ ID NO 45 YHCGSDHGSGNNYVYVFGGGTKLTVL SEQ ID NO. 46D11 QVQLQQWGAGLLKPSETLXXTCAVYGGSFSGYYWS QSALTQPPSVSAAPGQKVTISCSGSSSNIGNWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISV NYVSWYQQLPGTAPKLLIYDNNKRPSGIPDDTSKNQFSLQLNSVTPEDTAVYYCVRDDGMGLDAL RFSGSKSGTSATLGITGLQTGDEADYYCGTDIWGQGTMVTVSS SEQ ID NO 47 WDSSLSAEVFGGGTKVTVL SEQ ID NO. 48 E04EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGW LPVLTQPPSASGSPGQSVTISCTGTSSDVGGVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTIS YNYVSWYQQHPGKAPKLMIYEVSKRPSGVADKSISTAYLQWSSLKASDTAMYYCAGGGYYSYWG PDRFSGSKSGNTASLTVSGLQAEDEADYYCSQGTLVTVSS SEQ ID NO 49 SYAGSNNPYVFGTGTKVTVL SEQ ID NO. 50 E05QVQLVESGGGLIQPGGSVKLSCAASGFTVSSNYMS QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAWVRQAPGKGLEWVAVISYDGSFEYYGESVKGRFTIS GYDVHWYQQLPGTAPKLLIYGNSNRPSGVPRDNAKNTLYLQMSSLRPEDTAVYYCAKDTPYYYDSS DRFSGSKSGNTASLTVSGLQAEDEADYYCASGQSGDYFDHWGQGALVTVSS SEQ ID NO 51 WDDSLNSPVFGGGTKLTVL SEQ ID NO. 52 E08EVQLVESGGGLIQPGGSLRLSCAASGFTVSSNYMS QSVLTQPASVSGSPGQSITISCTGTSSDVGGWVRQAPGKGLEWVSVIYSGGSTYYADSVKGRFTISR YNFVSWYQQHPGKAPKLMIYDVTNRPSGVDNSKNTLYLQMNSLRAEDTAVYYCARDFGVGAVDY SNRFSGSKSGNTASLTISGLQAEDEADYYCSWGQGTLVTVSS SEQ ID NO 53 SYTDSSTLDVVFGGGTKVTVL SEQ ID NO. 54 F10QLQLQESGPGLVKPSQTLSLTCTVSGGSINSAGYYW QSALTQPASVSGSPGQSITISCTGTTSDVGGTWIRQHPGKGLEWIGSIYYSGSTYYNPSLKSRVSMS YNYVSWYQQHPGKGPKLMIYDVSKRPSGVQDTSKNQFSLKLSSLTAADTAVYYCAREVGVVPEYM SGRFSGSKSGNTASLTISGLQAEDEADYYCSDVWGQGTTVTVSS SEQ ID NO 55 SYTSSSTLLFGGXTKLTVL SEQ ID NO. 56 G01QVQLVESGGGLVQPGRSLRLSCAASGFTFSSYGMH QAGLTQPRSVSGSPGQSVTISCTGTSSDVGWVRQAPGKGLEWVGFIRSETYGGTTQTAASVKGRF GYNYVSWYQQHPGKAPKLLIYDVNNRPSGSISRDDSQGIAYLQMDSLKTEDTAVYFCARSGRGDF VPDRFSGSKSGNTASLTISGLQAEDEADYYCWGQGTLVTVSS SEQ ID NO 57 SSYTSSTTQVFGGGTKVTVL SEQ ID NO. 58 H06QVQLQESGGGVVQPGRSLXLSCAASGFTFSSYAMH QAGLTQPPSASGSPGQSVTISCTGTSSDVGWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI GYNYVSWYQQHPGKAPKLMIYEVNKRPSGSRDNSKNTLYLQMNSLRAEDTAVYYCARDRWDVA IPDRFSGSKSDSTASLTISGLQAEDEANYYCISSTVEATDGMDVWGQGTTVTVSS SEQ ID NO 59 YTSSTTWVFGGGTQLTVL SEQ ID NO. 60

We claim:
 1. A recombinant anti-ErbB3 antibody comprising a heavy chainvariable domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO. 3, SEQ ID NO. 5, and SEQ ID NO. 7, andcomprising a light chain variable domain having an amino acid sequenceselected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 6 and SEQID. NO. 8, wherein the antibody is an IgG.
 2. The recombinant fullyhuman antibody of claim 1, wherein the heavy chain variable domain hasan amino acid sequence as set forth in SEQ ID NO: 3, and the light chainvariable domain has an amino acid sequence as set forth in SEQ ID NO: 4.3. The recombinant fully human antibody of claim 1, wherein the heavychain variable domain has an amino acid sequence as set forth in SEQ IDNO: 5, and the light chain variable domain has an amino acid sequence asset forth in SEQ ID NO:
 6. 4. The recombinant fully human antibody ofclaim 1, wherein the heavy chain variable domain has an amino acidsequence as set forth in SEQ ID NO: 7, and the light chain variabledomain has an amino acid sequence as set forth in SEQ ID NO: 8.